In nature, Thiol/disulfide cycle plays an important role in thiol-based redox regulation and maintaining protein structure. However, little is known about how oxygen mediates the thiol/disulfide cycle under protein confinement. The main challenge is lacking real-time analysis techniques to track individual thiol/disulfide cycles in a confined space with a controllable local oxygen environment.
Recently, Professor Long and Professor Ying’s group developed the advancing nanopore single-molecule measurement instruments and the analytical approaches for studying single-molecule reaction.In this study, they utilized a mutant aerolysin nanopore to monitor the reversible thiol/disulfide cycle inside the glove box with the home-designed high-bandwidth weak current measurement system. By combining experiments with kinetic calculations, they discovered that confined local O2 could induce proton-involved, oxygen-induced disulfide bond cleavage. The negatively charged neighboring microenvironment facilitates disulfide bond cleavage, which plays a vital role in redox signaling and reactions. This instrument is proved to accurately measure reaction processes in complex solutions, providing new insight into redox reactions in life processes.
At first, this work monitors the kinetic process of thiol/disulfide cycles in protein nanopores at different oxygen concentrations. The experimental results showed that there was no significant change in the formation rate of disulfide bonds, while its bond breaking rate significantly decreased as the dissolved oxygen concentration decreased from 6.3 mg/L to<0.1mg/L (+50 mV) (Figure 1). Kinetic calculations show that the rate constant of oxygen-mediated disulfide bond formation in the protein nanopore reactor is 106 times higher than that in the macroscopic solution system under dissolved oxygen concentration of 6.3mg/L, which is equivalent to the rate constant of thiol oxidation in thioredoxin. This result confirms that the protein-confined environment can effectively improve the chemical reaction rate.
Figure 1 Nanopore experiments of the K238C Ael reacting with GSH in an N2 glove box.
Furthermore, the reaction rates constant of the thiol-disulfide cycle under different voltages was obtained using this instrument. The kinetics data under the in situ protein confinement was obtained by fitting the rate constants of each step in the thiol-disulfide cycle at 0 mV. It was found that oxygen and protons participated synergistically in the cleavage process of disulfide bonds under protein confinement. A new oxygen-mediated disulfide bond cleavage reaction pathway was proposed, and the reaction order of this process was calculated at the single-molecule level (Figure 2). Under the guidance of this mechanism, a novel G234C Aerolysin nanopore reactor was constructed by removing negatively charged amino acids to the protein reaction site, which exhibited a nearly 40-fold decrease rate constant of oxygen-mediated disulfide bond cleavage (Figure 3).
Figure 2 Probing the oxygen-mediated thiol/disulfide cycles in protein confinement.
Figure 3 The dynamics of thiol/disulfide bond cycle inside the G234C Ael nanopore.
The nanopore single-molecule chemical reaction measurement device with a controllable reaction environment and the atmosphere is expected to accurately measure reaction processes in complex solution systems, providing a new method for revealing the principle of redox reactions in chemical reactions and life processes.